Cellulase enzymes are widely used to improve the appearance and softness of cellulose-containing fabrics. A widespread application of cellulase enzymes is to remove cotton fuzz and loose surface fibers in or on the fabric. This process is referred to as “depilling”, “biopolishing” and “biofinishing” and smoothes the surface of the fabric, which in turn improves its softness and appearance. Cellulase treatment also aids in the prevention of subsequent formation of fiber pills that make the garments appear worn. During depilling it is desirable to minimize strength loss of the fabric due to the hydrolytic action of the cellulases.
Another industrial application of cellulase enzymes is for treating denim fabrics so as to impart to them a “stone-washed” appearance. Such a process is known in the industry as “bio-stoning”. The term bio-stoning was adopted as pumice stones were traditionally used to treat the fabric. However, cellulases have largely replaced pumice stones in recent years. Bio-stoning is quite different from depilling: biostoning aims to remove colour from denim and control its re-deposition on the fabric while depilling aims to solely improve softness and appearance as in depilling.
Cellulase enzymes are a group of glycoside hydrolase enzymes that catalyze the hydrolysis of beta-1,4-glycosidic linkages in the cellulose polymer and often comprise a cellulose binding domain (CBD) and a catalytic domain. A region between these two domains known as a “linker” or “linker peptide” serves as a flexible spacer between the CBD and the catalytic domain. The catalytic domains of individual cellulase components are classified by both the Enzyme Commission (EC) and the Glycoside Hydrolase (GH) family systems. The Enzyme Commission distinguishes two classes of cellulases based on their preference for cleavage of internal beta-1, 4 linkages (endoglucanase or “EG”, EC 3.2.1.4) or the release of cellobiose from the reducing or non-reducing end of the cellulose polymer (cellobiohydrolases or “CBH”, EC 3.2.1. 91, sometimes also referred to as exoglucanases). In contrast, the GH family system distinguishes the catalytic domains of cellulase components based on the conservation of primary and secondary structure, as well as the stereochemistry of the catalytic reaction. The GH family designations for all known cellulase catalytic and binding domains is provided and continually updated through the Carbohydrate-Active EnZymes (CAZy) database (Cantarel et al., 2009, Nucleic Acids Res 37:D233-238) available at the URL: cazy.org. Cellulase enzymes may be found in a number of GH Families including, but not limited to, Families 5, 6, 7, 8, 9, 10, 12, 16, 18, 19, 26, 44, 45, 48, 51, 61 and 74. Further, cellulase in some of the larger GH Families may be grouped into subfamilies.
Under the GH system, all cellulase catalytic domains, whether CBH or EG enzymes, are designated by “Cel” followed by the GH Family number. For example, cellulase comprising catalytic domains belonging to GH Families, 5, 6 and 7 may be referred to as Cel5, Cel6 and Cel7 cellulases, cellobiohydrolases or endoglucanases or as Family 5, Family 6, and Family 7 cellulases, cellobiohydrolases or endoglucanases. Designations such as Cel5, Cel6 and Cel7 may be followed by the capital letters A, B, C and so forth, which indicate the order in which the enzymes of the same family were identified from the source organisms. Further, the Cel designations may be preceded by a genus, species identifier such as “Tr” for Trichoderma reesei. 
Fungi such as Trichoderma secrete a number of different cellulase enzymes (also referred to herein as an “enzyme mixture”, “cellulase mixture” or “cellulase enzyme mixture”) that are individually known as components. The more prevalent of these enzyme components include cellobiohydrolase (CBH), also called exo-1,4-beta-D-glucanases, endoglucanase (EG), or endo-1,4-beta-D-glucanases, and beta-glucosidase enzymes. This classification is based on the enzymes' substrate specificities, i.e. affinity towards the chain ends (exo), the interior of the glucose chain (endo), or glycosidic bonds of soluble cellooligosaccharides and cellobiose (beta-glucosidase).
Trichoderma reesei is a widely studied and industrially important fungus for the production of cellulases. It produces at least six genetically different cellulases: two cellobiohydrolases, referred to as Cel6A (SEQ ID NO:3) and Cel7A (SEQ ID NO:5), also known as CBHII and CBHI, and at least four endoglucanases Cel5A (SEQ ID NO:2), Cel7B (SEQ ID NO:6), Cel12A and Cel45A (SEQ ID NO:4), also known as EGII, EGI, EGIII and EGV, respectively. Some of these cellulase components have been identified as contributing to improvements in depilling, while others are seen as detrimental as they can cause fabric strength loss, as discussed below.
Efforts have been made to improve the properties of cellulase mixtures for textile depilling by increasing the proportion of endoglucanase components in the secreted enzyme mixture relative to the natural mixture. For instance, WO 92/17574 discloses an approach that involves adjusting the amounts of EG type components relative to CBH I type components (Cel7A) so that the protein weight ratio of the EG to CBH I type components is greater than 5:1. Cotton-containing fabrics treated with such compositions exhibited decreased strength loss during depilling treatment compared to fabrics containing greater amounts of CBH I type (Cel7A) components. In addition, EP 866 165 discloses enzyme compositions enriched in EG II (Cel5A), which exhibit improvements in depilling efficiency and reduced strength loss. In embodiments, the enzyme compositions contain a cellulase protein content of at least 95% EG II.
U.S. Pat. No. 6,162,782 discloses that reduced strength loss of cotton fabrics can be achieved with cellulase detergent compositions that contain one or more endoglucanase components and reduced levels (less than 5 wt %) of CBH I (Cel7A). In these studies, it was found that CBH I, and to a certain extent CBH II, played a role in strength loss. The EG-enriched detergent compositions were reportedto impart improvements in softening, washing and color restoration. Similarly, Miettinen-Oinonen et al. (Applied and Environmental Microbiology, 2002, 68(8):3956-3964) reported improvements in visual appearance and reductions in pilling tendency with EG II-enriched cellulase preparations devoid of CBH I and CBH II. Miettinen-Oinonen et al. (VTT Publications 550, 2004, Trichoderma reesei strains for production of cellulases for the textile industry) also report good depilling results with EG II-enriched cellulase preparations. The use of endoglucanases in textile applications is also discussed in WO 2004/053039, EP 495 258 and U.S. Pat. Nos. 6,001,639, 5,958,082 and 5,948,672.
Moreover, a number of groups have contemplated the use of Cel45 cellulases in depilling. WO 97/14804 discloses a neutral Cel45A cellulase (20 K cellulase) from Melanocarpus origin for use in the textile and detergent industry . . . . WO2010/076388 discloses the production and use of Geomyces or Fusarium Cel45 endoglucanases in denim washing and depilling. U.S. Publication No. 2007/0111278 discloses the use of STCE1. a Cel45 endoglucanase, derived from Staphylotrichum, in washing or depilling of cellulose-containing fabrics. U.S. Pat. No. 7,741,093 discloses fusion of the Melanocarpus Cel45 endoglucanase to a linker peptide of Trichoderma reesei CBH I and a cellulose binding domain for bio-stoning and biofinishing. The purpose of constructing such fusion proteins was to increase the size of the Melanocarpus Cel45A enzyme, thereby decreasing the ability of the enzyme to penetrate the fabric, which in turn reduces strength loss. Similar approaches with Cel45 endoglucansaes and other cellulase enzymes are disclosed in WO 2007/118935 and U.S. Pat. No. 7,256,032.
Other groups have focused on elucidating whether or not cellulase enzyme components synergize with one another. The identification of synergistic combinations of enzyme components that provide for enhanced depilling could be a step forward with respect to improving process economics. Such improvements may be achieved since less enzyme protein, which is costly, would be necessary to impart the desired depilling effect.
Heikinheimo and Buchert (Textile Research Journal, 2001, 71(8):672-677) investigated the depilling properties of Trichoderma reesei EG I and II and CBH I and II cellulase components alone and in combination. Treatment of cotton interlock fabric with EG II-based combinations with CBH I or CBH II resulted in favourable depilling properties. However, the investigators also reported decreased depilling activity for combinations of the two endoglucanases, EG I and EG II. That is, no endo-endo synergy between the cellulase components was observed.
Cavaco-Paulo and Almeida (Textile Chemist and Colorist, 1996, 28(6):28-32) observed a high activity of EGI and II-deleted Trichoderma reesei cellulase mixtures on cotton cellulose. The authors state therein that the effect may possibly be due to synergy between the two CBH components or the CBH components and residual EG III or EG V. In Cavaco-Paulo, Carbohydrate Polymers, 1998, 37:273-277, it was stated that minor EG components seem to cooperate with the CBHs, in a synergistic fashion, to fully hydrolyse cotton. However, no testing was carried out to examine which particular components exhibited synergism with one another.
U.S. Pat. No. 5,958,083 discloses binary cellulase enzyme mixtures for use in bio-stoning. The first component is a Family 5 endoglucanase derived from Bacillus or Clostridium, or Family 7 endoglucanase derived from Humicolainsolens. The second component is a mechanical abrading agent, and/or an abrading cellulase (to form localized variation in color density), which may be a Family 12 or a Family 45 cellulase with a cellulose binding domain. Although improved bio-stoning with low backstaining was obtained, the properties of these compositions in depilling assays were not investigated.
Miettinen-Oinonen et al. (Enzyme and Microbial Technology, 2004, 34:332-341) examined the effect of Family 45 enzymes in biostoning, alone or in combination with other cellulase components, including endoglucanases. However, the depilling properties of these enzyme compositions were not tested in these studies.
Synergy between cellulase components has been reported in the literature in the hydrolysis of cellulosic substrates, or complete degradation and conversion of cellulose into ultimately glucose (Gusakov et al., Biotechnology and Bioengineering, 2007, 97(5):1028-1038; Andersen et al, Enzyme and Microbial Technology, 2008, 42(4):362-370; Boisset al., Biotechnology and Bioengineering, 2001, 72(3):339-345; Igarashi et al., Applied and Environmental Microbiology, 2008, 74:5628-5634; and Zhang et al., Biotechnology and Bioengineering, 2004, 7(88):797-824). However, extended hydrolysis is generally not a desired effect in depilling, as it may result in destruction of the fabric or severe strength loss. Furthermore, as reported by Ramos et al. (Biocatalysis and Biotransformation, 2007, 25(1):35-42) hydrolysis may not correlate with biopolishing effects.
Despite these efforts, there is still a need for improved combinations of cellulase enzymes and compositions thereof that are more effective in depilling of cellulose-containing goods. In particular, there is a continuous need for more efficient cellulase enzyme mixtures to improve the process economics. The present invention aims to meet these needs.